Allan wrote:
> Hi Peter,
>> Thanks for your input. I don't think I have explained myself well
> enough.
>>> This cut site is in the gene insert, not the vector.
>> My gene at this point is in pUC19 yielding pMyfavoritegene (pMFG). I
> cut it with SgrAI to linearize and dephosphorylate and gel purify only
> the resultant 6.3 KB band. At this point I don't know how a
> supercoiled pUC19 can migrate at 6.3 KB with the linearize pMFG.
It quite probably doesn't. There could easily be only a tiny amount there,
below the level of detection on a gel. Just because you don't see a band
doesn't mean it's not there.
Then, when your ligation fails, the only thing in the mix that's capable of
generating transformants is the uncut, uninserted vector.
Peter